This application is focused on defining the classical Hodgkin's lymphoma (HL) cancer stem cell and using information gained from the last grant cycle to develop novel tumor markers and immunotherapy approaches. Evidence from in vitro studies with HL cell lines suggests that there are two populations of cells: a bulk population and a cancer stem cell population that is clonogenic and self-renewing; Some potential immunotherapy targets are expressed by both populations, while others appear to be expressed by only the bulk population or the clonogenic cancer stem cell population. In the first aim, we seek to better characterize HL cancer stem cells (phenotype, frequency, distribution) and to characterize distinctive aspects of their patterns of DNA methylation. Investigations use cell lines, murine xenografts and clinical specimens. In the second aim, we investigate plasma tumor markers (Ig gene rearrangement, patterns of DNA methylation, EBV DNA) that may be especially useful in the context of cancer stem cell-targeted therapy. In the third aim, we propose to augment standardtherapies with immunologic therapies in hopes of increasing the progression-free survival rate. This will involve two clinical trials. A trial is proposed in which vaccine is combined with rituximab (also targeting the HL cancer stem cell) in the context of high dose cyclophosphamide therapy. The vaccine was developed in the previous granting cycle. A hematopoietic tumor cell line (K562) that expresses several potential cancer stem cell antigens (survivin, telomerase) was engineered also to express EBV antigens (EBNA1, LMP2) and GM-CSF. The trial will evaluate failure-free survival as well as the ability to induce CD4 and CDS T cell responses using a cell-based allogeneic vaccine. Immune responses survivin, telomerase, EBV EBNA1 and EBV LMP2 will be assessed: Responses to EBV LMP2 are well characterized in healthy volunteers and patients with EBV(+) and EBV(-) HL. In addition to potentially stimulating anti-tumor responses in EBV(+)HL, the immune response will also serve as a reporter system for monitoring the effect of the vaccination strategy. This trial is aimed at patients with relapsed sensitive disease, has FDA and IRB approval and is beginning to accrue. In a second trial, newly diagnosed patients will be treated with ABVD and rituximab. Progression-free survival, the utility of the DNA tumor markers and the impact of therapy on cancer stem cells in the periphery will be assessed.